Functional influenza virus-like particles (VLPS)

ABSTRACT

Recombinant influenza virus proteins, including influenza capsomers, subviral particles, virus-like particles (VLP), VLP complexes, and/or any portions of thereof, are provided as a vaccine for influenza viruses. The invention is based on the combination of two vaccine technologies: (1) intrinsically safe recombinant vaccine technology, and (2) highly immunogenic, self-assembled protein macromolecules embedded in plasma membranes and comprised of multiple copies of influenza virus structural proteins exhibiting neutralizing epitopes in native conformations. More specifically, this invention relates to the design and production of functional homotypic and heterotypic recombinant influenza virus-like particles (VLPs) comprised of recombinant structural proteins of human influenza virus type A/Sydney/5/94 (H3N2) and/or avian influenza virus type A/Hong Kong/1073/99 (H9N2) in baculovirus-infected insect cells and their application as a vaccine in the prevention of influenza infections and as a laboratory reagent for virus structural studies and clinical diagnostics.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 13/796,125, filedMar. 12, 2013, which is a continuation of U.S. Ser. No. 10/617,569 filedJul. 11, 2003, now U.S. Pat. No. 8,592,197, each of which isincorporated herein in its entirety for all purposes.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:“NOVV_003_02_US_SeqList.txt” date recorded: Mar. 11, 2013, file size 9kb).

BACKGROUND OF INVENTION

Influenza virus is a member of Orthomyxoviridae family (for review, seeMurphy and Webster, 1996). There are three subtypes of influenza virusesdesignated A, B, and C. The influenza virion contains a segmentednegative-sense RNA genome. The influenza virion includes the followingproteins: hemagglutinin (HA), neuraminidase (NA), matrix (M1), protonion-channel protein (M2), nucleoprotein (NP), polymerase basic protein 1(PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), and nonstructural protein 2 (NS2) proteins. The HA, NA, M1, and M2are membrane associated, whereas NP, PB 1, PB2, P A, and NS2 arenucleocapsid associated proteins. The NS 1 is the only nonstructuralprotein not associated with virion particles but specific forinfluenza-infected cells. The M1 protein is the most abundant protein ininfluenza particles. The HA and NA proteins are envelope glycoproteins,responsible for virus attachment and penetration of the viral particlesinto the cell, and the sources of the major immunodominant epitopes forvirus neutralization and protective immunity. Both HA and NA proteinsare considered the most important components for prophylactic influenzavaccines.

Influenza virus infection is initiated by the attachment of the virionsurface HA protein to a sialic acid-containing cellular receptor(glycoproteins and glycolipids). The NA protein mediates processing ofthe sialic acid receptor, and virus penetration into the cell depends onHA-dependent receptor-mediated endocytosis. In the acidic confines ofinternalized endosomes containing an influenza virion, the HA₂ proteinundergoes conformational changes that lead to fusion of viral and cellmembranes and virus uncoating and M2-mediated release of M1 proteinsfrom nucleocapsid-associated ribonucleoproteins (RNPs), which migrateinto the cell nucleus for viral RNA synthesis. Antibodies to HA proteinsprevent virus infection by neutralizing virus infectivity, whereasantibodies to NA proteins mediate their effect on the early steps ofviral replication.

Inactivated influenza A and B virus vaccines are licensed currently forparenteral administration. These trivalent vaccines are produced in theallantoic cavity of embryonated chick eggs, purified by rate zonalcentrifugation or column chromatography, inactivated with formalin orβ-propiolactone, and formulated as a blend of the two strains of type Aand the type B strain of influenza viruses in circulation among thehuman population for a given year. The available commercial influenzavaccines are whole virus (WV) or subvirion (SV; split or purifiedsurface antigen) virus vaccines. The WV vaccine contains intact,inactivated virions. SV vaccines treated with solvents such astri-n-butyl phosphate (Flu-Shield, Wyeth-Lederle) contain nearly all ofthe viral structural proteins and some of the viral envelopes. SVvaccines solubilized with Triton X-100 (Fluzone, Connaught; Fluvirin,Evans) contain aggregates of HA monomers, NA, and NP principally,although residual amounts of other viral structural proteins arepresent. A potential cold-adapted live attenuated influenza virusvaccine (FluMist, MedImmune) was granted marketing approval recently bythe FDA for commercial usage as an intranasally delivered vaccineindicated for active immunization and the prevention of disease causedby influenza A and B viruses in healthy children and adolescents, 5-17years of age and healthy adults 18-49 years of age.

Several recombinant products have been developed as recombinantinfluenza vaccine candidates. These approaches have focused on theexpression, production, and purification of influenza type A HA and NAproteins, including expression of these proteins using baculovirusinfected insect cells (Crawford et al, 1999; Johansson, 1999; Treanor etal., 1996), viral vectors (Pushko et al., 1997; Berglund et al, 1999),and DNA vaccine constructs (Olsen et al., 1997).

Crawford et al. (1999) demonstrated that influenza HA expressed inbaculovirus infected insect cells is capable of preventing lethalinfluenza disease caused by avian H5 and H7 influenza subtypes. At thesame time, another group demonstrated that baculovirus-expressedinfluenza HA and NA proteins induce immune responses in animals superiorto those induced by a conventional vaccine (Johansson et al., 1999).Immunogenicity and efficacy of baculovirus-expressed hemagglutinin ofequine influenza virus was compared to a homologous DNA vaccinecandidate (Olsen et al., 1997). Taken together, the data demonstratedthat a high degree of protection against influenza virus challenge canbe induced with recombinant HA or NA proteins, using variousexperimental approaches and in different animal models.

Lakey et al. (1996) showed that a baculovirus-derived influenza HAvaccine was well-tolerated and immunogenic in human volunteers in aPhase I dose escalation safety study. However, results from Phase IIstudies conducted at several clinical sites in human volunteersvaccinated with several doses of influenza vaccines comprised of HAand/or NA proteins indicated that the recombinant subunit proteinvaccines did not elicit protective immunity [G. Smith, Protein Sciences;M. Perdue, USDA, Personal Communications]. These results indicated thatconformational epitopes displayed on the surface of HA and NA peplomersof infectious virions were important in the elicitation of neutralizingantibodies and protective immunity.

Regarding the inclusion of other influenza proteins in recombinantinfluenza vaccine candidates, a number of studies have been carried out,including the experiments involving influenza nucleoprotein, NP, aloneor in combination with M1 protein (Ulmer et al., 1993; Ulmer et al.,1998; Zhou et al., 1995; Tsui et al., 1998). These vaccine candidates,which were composed of quasi-invariant inner virion proteins, elicited abroad spectrum immunity that was primarily cellular (both CD4⁺ and CD8⁺memory T cells). These experiments involved the use of the DNA or viralgenetic vectors. Relatively large amounts of injected DNA were needed,as results from experiments with lower doses of DNA indicated little orno protection (Chen et al., 1998). Hence, further preclinical andclinical research may be required to evaluate whether such DNA-basedapproaches involving influenza NP and M1 are safe, effective, andpersistent.

Recently, in an attempt to develop more effective vaccines forinfluenza, particulate proteins were used as carriers of influenza M2protein epitopes. The rationale for development of an M2-based vaccinewas that in animal studies protective immunity against influenza waselicited by M2 proteins (Slepushkin et al., 1995). Neirynck et al.(1999) used a 23-aa long M2 transmembrane domain as an amino terminalfusion partner with the hepatitis B virus core antigen (HBcAg) to exposethe M2 epitope(s) on the surface of HBcAg capsid-like particles.However, in spite of the fact that both full-length M2 protein andM2-HBcAg VIP induced detectable antibodies and protection in mice, itwas unlikely that future influenza vaccines would be based exclusivelyon the M2 protein, as the M2 protein was present at low copy number pervirion, was weakly antigenic, was unable to elicit antibodies that boundfree influenza virions, and was unable to block virus attachment to cellreceptors (i.e. virus neutralization).

Since previous research has shown that the surface influenzaglycoproteins, HA and NA, are the primary targets for elicitation ofprotective immunity against influenza virus and that M1 provides aconserved target for cellular immunity to influenza, a new vaccinecandidate may include these viral antigens as a protein macromolecularparticle, such as virus-like particles (VLPs). Further, the particlewith these influenza antigens may display conformational epitopes thatelicit neutralizing antibodies to multiple strains of influenza viruses.

Several studies have demonstrated that recombinant influenza proteinscould self-assemble into VLPs in cell culture using mammalian expressionplasmids or baculovirus vectors (Gomez-Puertas et al., 1999; Neumann etal., 2000; Latham and Galarza, 2001). Gomez-Puertas et al. (1999)demonstrated that efficient formation of influenza VLP depends on theexpression levels of viral proteins. Neumann et al. (2000) established amammalian expression plasmid-based system for generating infectiousinfluenza virus-like particles entirely from cloned cDNAs. Latham andGalarza (2001) reported the formation of influenza VLPs in insect cellsinfected with recombinant baculovirus co-expressing HA, NA, M1, and M2genes. These studies demonstrated that influenza virion proteins mayself-assemble upon co-expression in eukaryotic cells.

SUMMARY OF INVENTION

According to the present invention, macromolecular protein structuresare provided that comprise avian influenza virus type A H9N2 codingsequences for HA (GenBank Accession No. AJ404626), NA (GenBank AccessionNo. AJ404629), M1 (GenBank Accession No. AJ278646), M2 (GenBankAccession No. AF255363), and NP (GenBank Accession No. AF255742)proteins and that comprise human influenza virus type A H3N2 codingsequences for HA (GenBank Accession No. AJ311466) and for NA (GenBankAccession No. AJ291403). The genomic RNA encoding these influenza viralgenes may be isolated from influenza virus isolates or from tissues ofinfluenza-infected organisms. Each of these coding sequences from thesame or different strains or types of influenza virus is cloneddownstream of transcriptional promoters within expression vectors andare expressed in cells.

Thus, the invention provides a macromolecular protein structurecontaining (a) a first influenza virus M1 protein and (b) an additionalstructural protein, which may include a second or more influenza virusM1 protein; a first, second or more influenza virus HA protein; a first,second, or more influenza virus NA protein; and a first, second, or moreinfluenza virus M2 protein. If the additional structural protein is notfrom a second or more influenza virus M1 protein, then both or allmembers of the group, e.g., first and second influenza M2 virus proteinsare included. As such, there is provided a functional influenza proteinstructure, including a subviral particle, VLP, or capsomer structure, ora portion thereof, a vaccine, a multivalent vaccine, and mixturesthereof consisting essentially of influenza virus structural proteinsproduced by the method of the invention. In a particularly preferredembodiment, the influenza macromolecular protein structure includesinfluenza virus HA, NA, and M1 proteins that are the expression productsof influenza virus genes cloned as synthetic fragments from a wild typevirus.

The macromolecular protein structure may also include an additionalstructural protein, for example, a nucleoprotein (NP), membrane proteinsfrom species other than noninfluenza viruses and a membrane protein froma non-influenza source, which are derived from avian or mammalianorigins and different subtypes of influenza virus, including subtype Aand B influenza viruses. The invention may include a chimericmacromolecular protein structure, which includes a portion of at leastone protein having a moiety not produced by influenza virus.

Prevention of influenza may be accomplished by providing amacromolecular protein structure that may be self-assembled in a hostcell from a recombinant construct. The macromolecular protein structureof the invention has the ability to self-assemble into homotypic orheterotypic virus-like particles (VLPs) that display conformationalepitopes on HA and NA proteins, which elicit neutralizing antibodiesthat are protective. The composition may be a vaccine composition, whichalso contains a carrier or diluent and/or an adjuvant. The functionalinfluenza VLPs elicit neutralizing antibodies against one or morestrains or types of influenza virus depending on whether the functionalinfluenza VLPs contain HA and/or NA proteins from one or more viralstrains or types. The vaccine may include influenza virus proteins thatare wild type influenza virus proteins. Preferably, the structuralproteins containing the influenza VLP, or a portion of thereof, may bederived from the various strains of wild type influenza viruses. Theinfluenza vaccines may be administered to humans or animals to elicitprotective immunity against one or more strains or types of influenzavirus.

The macromolecular protein structures of the invention may exhibithemagglutinin activity and/or neuraminidase activity.

The invention provides a method for producing a VLP derived frominfluenza by constructing a recombinant construct that encodes influenzastructural genes, including M1, HA, and at least one structural proteinderived from influenza virus. A recombinant construct is used totransfect, infect, or transform a suitable host cell with therecombinant baculovirus. The host cell is cultured under conditionswhich permit the expression of M1, HA and at least one structuralprotein derived from influenza virus and the VLP is formed in the hostcell. The infected cell media containing a functional influenza VLP isharvested and the VLP is purified. The invention also features anadditional step of co-transfecting, co-infecting or co-transforming thehost cell with a second recombinant construct which encodes a secondinfluenza protein, thereby incorporating the second influenza proteinwithin the VLP. Such structural proteins may be derived from influenzavirus, including NA, M2, and NP, and at least one structural protein isderived from avian or mammalian origins. The structural protein may be asubtype A and B influenza viruses. According to the invention, the hostcell may be a eukaryotic cell. In addition, the VLP may be a chimericVLP.

The invention also features a method of formulating a drug substancecontaining an influenza VLP by introducing recombinant constructsencoding influenza viral genes into host cells and allowingself-assembly of the recombinant influenza viral proteins into afunctional homotypic or heterotypic VLP in cells. The influenza VLP isisolated and purified and a drug substance is formulated containing theinfluenza VLP. The drug substance may further include an adjuvant. Inaddition, the invention provides a method for formulating a drugproduct, by mixing such a drug substance containing an influenza VLPwith a lipid vesicle, i.e., a non-ionic lipid vesicle. Thus, functionalhomotypic or heterotypic VLPs may bud as enveloped particles from theinfected cells. The budded influenza VLPs may be isolated and purifiedby ultracentrifugation or column chromatography as drug substances andformulated alone or with adjuvants such as Novasomes®, a product ofNovavax, Inc., as drug products such as vaccines. Novasomes®, whichprovide an enhanced immunological effect, are further described in U.S.Pat. No. 4,911,928, which is incorporated herein by reference.

The invention provides a method for detecting humoral immunity toinfluenza virus infection in a vertebrate by providing a test reagentincluding an effective antibody-detecting amount of influenza virusprotein having at least one conformational epitope of an influenza virusmacromolecular structure. The test reagent is contacted with a sample ofbodily fluid from a vertebrate to be examined for influenza virusinfection. Influenza virus specific antibodies contained in the sampleare allowed to bind to the conformational epitope of an influenza virusmacromolecular structure to form antigen-antibody complexes. Thecomplexes are separated from unbound complexes and contacted with adetectably labeled immunoglobulin-binding agent. The amount of thedetectably labeled immunoglobulin-binding agent that is bound to thecomplexes is determined.

Influenza virus may be detected in a specimen from an animal or humansuspected of being infected with the virus by providing antibodies,which have a detectable signal producing label, or are attached to adetectably labeled reagent, having specificity to at least oneconformational epitope of the particle of the influenza virus. Thespecimen is contacted with antibodies and the antibodies are allowed tobind to the influenza virus. The presence of influenza virus in thespecimen is determined by means of the detectable label.

The invention provides methods for treatment, prevention, and generatinga protective immune response by administering to a vertebrate aneffective amount of the composition of the invention.

Alternatively, the influenza VLP drug substance may be formulated aslaboratory reagents used for influenza virus structure studies andclinical diagnostic assays. The invention also provides a kit fortreating influenza virus by administering an effective amount of acomposition of the invention and directions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the nucleotide sequence of avian influenza A/HongKong/1073/99 (H9N2) virus neuraminidase (NA) gene (SEQ ID NO:1).

FIG. 2 depicts the nucleotide sequence of avian influenza A/HongKong/1073/99 (H9N2) virus hemagglutinin (HA) gene (SEQ ID NO:2).

FIG. 3 depicts the nucleotide sequence of avian influenza A/HongKong/1073/99 (H9N2) virus matrix protein M1 (M1) gene (SEQ ID NO:3).

FIG. 4 depicts the transfer vectors for construction of recombinantbaculoviruses for expression of avian influenza A/Hong Kong/1073/99(H9N2) HA, NA, and M1 proteins. FIG. 4A depicts a transfer vector forexpression of individual genes and FIG. 4B depicts the transfer vectorfor multi-expression of the genes.

FIG. 5 depicts the expression of avian influenza A/Hong Kong/1073/99(H9N2) virus HA, NA, and M1 proteins in Sf-9S cells.

FIG. 6 depicts the purification of avian influenza A/Hong Kong/1073/99(H9N2) VLPs by the sucrose density gradient method.

FIG. 7 depicts the detection of influenza virus protein by gelfiltration chromatography. The antibodies used in the Western blotanalyses are as follows: (A) rabbit anti-H9N2; (b) murine anti-M1 mAb;and (C) murine anti-BACgp64.

FIG. 8 depicts the detection of avian influenza A/Hong Kong/1073/99(H9N2) proteins including subviral particles, VLP, and VLP complexes, byelectron microscopy.

FIG. 9 depicts the hemagglutination activity of purified avian influenzaA/Hong Kong/1073/99 (H9N2) VLPs.

FIG. 10 depicts the neuraminidase activity of purified avian influenzaA/Hong Kong/1073/99 (H9N2) VLPs.

FIG. 11 depicts the immunization and bleed schedule for theimmunogenicity study of recombinant influenza with purified avianinfluenza A/Hong Kong/1073/99 (H9N2) VLPs in mice.

FIG. 12 depicts the results of an immunogenicity study in mice immunizedwith recombinant influenza H9N2 VLPs. FIG. 12A depicts sera from BALB/cmice immunized with recombinant VLPs comprised of HA, NA, and M1proteins from avian influenza virus type A/H9N2/Hong Kong/1073/99. FIG.12B depicts sera from New Zealand white rabbits immunized withinactivated avian influenza virus type A H9N2 were reacted with Westernblots containing inactivated avian influenza virus type A H9N2 (lanes 1and 3) or cold-adapted avian influenza virus type A H9N2 (lanes 2 and4).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “baculovius,” also known as baculoviridae,refers to a family of enveloped DNA viruses of arthropods, members ofwhich may be used as expression vectors for producing recombinantproteins in insert cell cultures. The virion contains one or morerod-shaped nucleocapsids containing a molecule of circular supercoileddouble-stranded DNA (M_(r) 54×10⁶-154×10⁶). The virus used as a vectoris generally Autographa californica nuclear polyhedrosis virus (NVP).Expression of introduced genes is under the control of the strongpromoter that normally regulates expression of the polyhedron proteincomponent of the large nuclear inclusion in which the viruses areembedded in the infected cells.

As used herein, the term “derived from” refers to the origin or source,and may include naturally occurring, recombinant, unpurified, orpurified molecules. The proteins and molecules of the present inventionmay be derived from influenza or non-influenza molecules.

As used herein the term “first” influenza virus protein, i.e., a firstinfluenza virus M1 protein, refers to a protein, such as M1, HA, NA, andM2, that is derived from a particular strain of influenza virus. Thestrain or type of the first influenza virus differs from the strain ortype of the second influenza virus protein. Thus, “second” influenzavirus protein, i.e., the second influenza virus M1 protein, refers to aprotein, such as M1, HA, NA, and M2, that is derived from a secondstrain of influenza virus, which is a different strain or type than thefirst influenza virus protein.

As used herein, the term “hemagglutinin activity” refers to the abilityof HA-containing proteins, VLPs, or portions thereof to bind andagglutinate red blood cells (erythrocytes).

As used herein, the term “neuraminidase activity” refers to theenzymatic activity of NA-containing proteins, VLPs, or portions thereofto cleave sialic acid residues from substrates including proteins suchas fetuin.

As used herein, the term “heterotypic” refers to one or more differenttypes or strains of virus.

As used herein, the term “homotypic” refers to one type or strain ofvirus.

As used herein, the term “macromolecular protein structure” refers tothe construction or arrangement of one or more proteins.

As used herein, the term “multivalent” vaccine refers to a vaccineagainst multiple types or strains of influenza virus.

As used herein, the term “non-influenza” refers to a protein or moleculethat is not derived from influenza virus.

As used herein, the term “vaccine” refers to a preparation of dead orweakened pathogens, or of derived antigenic determinants, that is usedto induce formation of antibodies or immunity against the pathogen. Avaccine given to provide immunity to the disease, for example,influenza, which is caused by influenza viruses. The present inventionprovides vaccine compositions that are immunogenic and provideprotection.

Influenza remains a pervasive public health concern despite theavailability of specific inactivated virus vaccines that are 60-80%effective under optimal conditions. When these vaccines are effective,illness is usually averted by preventing viral infection. Vaccinefailure can occur as a result of accumulated antigenic differences(antigenic shift and antigenic drift). For example, avian influenzavirus type A H9N2 co-circulated with human influenza virus type ASydney/97 H3N2 in pigs and led to genetic reassortment and emergence ofnew strains of human influenza virus with pandemic potential (Peiris etal., 2001). In the event of such antigenic shift, it is unlikely thatcurrent vaccines would provide adequate protection.

Another reason for the paucity of influenza vaccine programs is therelatively short persistence of immunity elicited by the currentvaccines. Further inadequacy of influenza control measures reflectsrestricted use of current vaccines because of vaccine reactogenicity andside effects in young children, elderly, and people with allergies tocomponents of eggs, which are used in manufacturing of commerciallylicensed inactivated virus influenza vaccines.

Additionally, inactivated influenza virus vaccines often lack or containaltered HA and NA conformational epitopes, which elicit neutralizingantibodies and play a major role in protection against disease. Thus,inactivated viral vaccines, as well as some recombinant monomericinfluenza subunit protein vaccines, deliver inadequate protection. Onthe other hand, macromolecular protein structures, such as capsomers,subviral particles, and/or VLPs, include multiple copies of nativeproteins exhibiting conformational epitopes, which are advantageous foroptimal vaccine immunogenicity.

The present invention describes the cloning of avian influenza A/HongKong/1073/99 (H9N2) virus HA, NA, and M1 genes into a single baculovirusexpression vector alone or in tandem and production of influenza vaccinecandidates or reagents comprised of recombinant influenza structuralproteins that self-assemble into functional and immunogenic homotypicmacromolecular protein structures, including subviral influenzaparticles and influenza VLP, in baculovirus-infected insect cells.

The present invention further features the cloning of human influenzaA/Sydney/5/94 (H3N2) virus HA, NA, M1, M2, and NP genes into baculovirusexpression vectors and production influenza vaccine candidates orreagents comprised of influenza structural proteins that self-assembleinto functional and immunogenic homotypic macromolecular proteinstructures, including subviral influenza particles and influenza VLP, inbaculovirus-infected insect cells.

In addition, the instant invention describes the cloning of the HA geneof human influenza A/Sydney/5/94 (H3N2) virus and the HA, NA, and M1genes of avian influenza A/Hong Kong/1073/99 (H9N2) into a singlebaculovirus expression vector in tandem and production influenza vaccinecandidates or reagents comprised of influenza structural proteins thatself-assemble into functional and immunogenic heterotypic macromolecularprotein structures, including subviral influenza particles and influenzaVLP, in baculovirus-infected insect cells.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures and the Sequence Listing, areincorporated herein by reference.

SPECIFIC EXAMPLES Example 1 Materials and Methods

Avian influenza A/Hong Kong/1073/99 (H9N2) virus HA, NA, and M1 geneswere expressed in Spodoptera frugiperda cells (Sf-9S cell line; ATCCPTA-4047) using the baculovirus bacmid expression system. The HA, NA,and M1 genes were synthesized by the reverse transcription andpolymerase chain reaction (PCR) using RNA isolated from avian influenzaA/Hong Kong/1073/99 (H9N2) virus (FIGS. 1, 2, and 3). For reversetranscription and PCR, oligonucleotide primers specific for avianinfluenza A/Hong Kong/1073/99 (H9N2) virus HA, NA, and M1 genes wereused (Table 1). The cDNA copies of these genes were cloned initiallyinto the bacterial subcloning vector, pCR2.1TOPO. From the resultingthree pCR2.1TOPO-based plasmids, the HA, NA, and M1 genes were inserteddownstream of the AcMNPV polyhedrin promoters in the baculovirustransfer vector, pFastBac1 (InVitrogen), resulting in threepFastBac1-based plasmids: pHA, pNA, and pM1 expressing these influenzavirus genes, respectively. Then, a single pFastBac1-based plasmid pHAMwas constructed encoding both the HA and M1 genes, each downstream froma separate polyhedrin promoter (FIG. 4). The nucleotide sequence of theNA gene with the adjacent 5′- and 3′-regions within the pNA plasmid wasdetermined (SEQ ID NO:1) (FIG. 1). At the same time, the nucleotidesequences of the HA and M1 genes with the adjacent regions were alsodetermined using the pHAM plasmid (SEQ ID NOS:2 and 3) (FIGS. 2 and 3).

Finally, a restriction DNA fragment from the pHAM plasmid that encodedboth the HA and M1 expression cassettes was cloned into the pNA plasmid.This resulted in the plasmid pNAHAM encoding avian influenza A/HongKong/1073/99 (H9N2) virus HA, NA, and M1 genes (FIG. 4).

Plasmid pNAHAM was used to construct a recombinant baculoviruscontaining influenza virus NA, HA, and M1 genes integrated into thegenome, each downstream from a separate baculovirus polyhedrin promoter.Infection of permissive Sf-9S insect cells with the resultingrecombinant baculovirus resulted in co-expression of these threeinfluenza genes in each Sf-9S cell infected with such recombinantbaculovirus.

Results

The expression products in infected Sf-9S cells were characterized at 72hr postinfection (p.i.) by SDS-PAGE analysis, Coomassie blue proteinstaining, and Western immunoblot analysis using HA- and M1-specificantibodies (FIG. 5). Western immunoblot analysis was carried out usingrabbit antibody raised against influenza virus type A/Hong Kong/1073/99(H9N2) (CDC, Atlanta, Ga., USA), or mouse monoclonal antibody toinfluenza M1 protein (Serotec, UK). The HA, NA, and M1 proteins of theexpected molecular weights (64 kd, 60 kd, and 31 kd, respectively) weredetected by Western immunoblot analysis. Compared to the amount of HAprotein detected in this assay, the NA protein showed lower reactivitywith rabbit serum to influenza A/Hong Kong/1073/99 (H9N2) virus.Explanations for the amount of detectable NA protein included lowerexpression levels of the NA protein from Sf-9S cells infected withrecombinant baculovirus as compared to the HA protein, lower reactivityof the NA with this serum under denaturing conditions in the Westernimmunoblot assay (due to the elimination of important NA epitopes duringgel electrophoresis of membrane binding), lower NA-antibody avidity ascompared to HA-antibody, or a lower abundance of NA-antibodies in theserum.

The culture medium from the Sf-9S cells infected with recombinantbaculovirus expressing A/Hong Kong/1073/99 (H9N2) HA, NA, and M1proteins was also probed for influenza proteins. The clarified culturesupernatants were subjected to ultracentrifugation at 27,000 rpm inorder to concentrate high-molecular protein complexes of influenzavirus, such as subviral particles, VLP, complexes of VLP, and possibly,other self-assembled particulates comprised of influenza HA, NA, and M1proteins. Pelleted protein products were resuspended inphosphate-buffered saline (PBS, pH 7.2) and further purified byultracentrifugation on discontinuous 20-60% sucrose step gradients.Fractions from the sucrose gradients were collected and analyzed bySDS-PAGE analysis, Western immunoblot analysis, and electron microscopy.

Influenza HA and M1 proteins of the expected molecular weights weredetected in multiple sucrose density gradient fractions by Coomassieblue staining and Western immunoblot analysis (FIG. 6). This suggestedthat influenza viral proteins from infected Sf-9S cells are aggregatedin complexes of high-molecular weight, such as capsomers, subviralparticles, VLP, and/or VLP complexes. The NA proteins, althoughinconsistently detected by Coomassie blue staining and Westernimmunoblot analysis, which was likely due to the inability of the rabbitanti-influenza serum to recognize denatured NA protein in the Westernimmunoblot assay, were consistently detected in neuraminidase enzymeactivity assay (FIG. 10).

The presence of high-molecular VLPs was confirmed by gel filtrationchromatography. An aliquot from sucrose density gradient fractionscontaining influenza viral proteins was loaded onto a Sepharose CL-4Bcolumn for fractionation based on mass. The column was calibrated withdextran blue 2000, dextran yellow, and vitamin B12 (Amersham Pharmacia)with apparent molecular weights of 2,000,000; 20,000; and 1,357 daltons,respectively, and the void volume of the column was determined. Asexpected, high-molecular influenza viral proteins migrated in the voidvolume of the column, which was characteristic of macromolecularproteins, such as virus particles. Fractions were analyzed by Westernimmunoblot analysis to detect influenza and baculovirus proteins. Forexample, M1 proteins were detected in the void volume fractions, whichalso contained baculovirus proteins (FIG. 7).

The morphology of influenza VLPs and proteins in sucrose gradientfractions was elucidated by electron microscopy. For negative-stainingelectron microscopy, influenza proteins from two sucrose densitygradient fractions were fixed with 2% glutaraldehyde in PBS, pH 7.2.Electron microscopic examination of negatively-stained samples revealedthe presence of macromolecular protein complexes or VLPs in bothfractions. These VLPs displayed different sizes including diameters ofapproximately 60 and 80 nm and morphologies (spheres). Larger complexesof both types of particles were also detected, as well as rod-shapedparticles (FIG. 8). All observed macromolecular structures had spikes(peplomers) on their surfaces, which is characteristic of influenzaviruses. Since the size and appearance of 80 nm particles was similar toparticles of wild type influenza virus, these structures likelyrepresented VLPs, which have distinct similarities to wild typeinfluenza virions, including similar particle geometry, architecture,triangulation number, symmetry, and other characteristics. The smallerparticles of approximately 60 nm may represent subviral particles thatdiffer from VLPs both morphologically and structurally. Similarphenomenon of recombinant macromolecular proteins of different sizes andmorphologies was also reported for other viruses. For example,recombinant core antigen (HBcAg) of hepatitis B virus forms particles ofdifferent sizes, which have different architecture and triangulationnumber T=4 and T=3, respectively (Crowther et al., 1994).

To characterize the functional properties of the purified influenzaA/Hong Kong/1073/99 (H9N2) VLPs, samples were tested in ahemagglutination assay (FIG. 9) and a neuraminidase enzyme assay (FIG.10). For the hemagglutination assay, 2-fold dilutions of purifiedinfluenza VLPs were mixed with 0.6% guinea pig red blood cells andincubated at 4° C. for 1 hr or 16 hr. The extent of hemagglutination wasinspected visually and the highest dilution of recombinant influenzaproteins capable of agglutinating red blood cells was determined andrecorded (FIG. 9). Again, many fractions from the sucrose densitygradient exhibited hemagglutination activity, suggesting that multiplemacromolecular and monomeric forms of influenza proteins were present.The highest titer detected was 1:4000. In a control experiment,wild-type influenza A/Shangdong virus demonstrated a titer of 1:2000.The hemagglutination assay revealed that the recombinant VLPs consistingof influenza A/Hong Kong/1073/99 (H9N2) virus HA, NA, and M1 proteinswere functionally active. This suggested that the assembly,conformation, and folding of the HA subunit proteins within the VLPswere similar or identical to that of the wild type influenza virus.

Additionally, a neuraminidase enzyme assay was performed on samples ofpurified H9N2 VLPs. The amount of neuraminidase activity in sucrosedensity gradient fractions was determined using fetuin as a substrate.In the neuraminidase assay, the neuraminidase cleaved sialic acid fromsubstrate molecules to release sialic acid for measurement. Arsenitereagent was added to stop enzyme activity. The amount of sialic acidliberated was determined chemically with thiobarbituric acid thatproduces a pink color that was proportional to the amount of free sialicacid. The amount of color (chromophor) was measuredspectrophotometrically at wavelength 549 nm. Using this method,neuraminidase activity was demonstrated in sucrose gradient fractionscontaining influenza VLPs (FIG. 10). As expected, the activity wasobserved in several fractions, with two peak fractions. As a positivecontrol, wild type influenza virus was used. The wild type influenzavirus exhibited neuraminidase enzyme activity comparable to that ofpurified influenza VLPs. These findings corroborated the HA results withregard to protein conformation and suggested that purified VLPs ofinfluenza A/Hong Kong/1073/99 (H9N2) virus were functionally similar towild type influenza virus.

The results from the above analyses and assays indicated that expressionof influenza A/Hong Kong/1073/99 (H9N2) HA, NA, and M1 proteins wassufficient for the self-assembly and transport of functional VLPs frombaculovirus-infected insect cells. Since these influenza VLPsrepresented self-assembled influenza structural proteins anddemonstrated functional and biochemical properties similar to those ofwild type influenza virus, these influenza VLPs conserved importantstructural conformations including surface epitopes necessary foreffective influenza vaccines.

Example 2 RT-PCR Cloning of Avian Influenza A/Hong Kong/1073/99 ViralGenes

It is an object of the present invention to provide synthetic nucleicacid sequences capable of directing production of recombinant influenzavirus proteins. Such synthetic nucleic acid sequences were obtained byreverse transcription and polymerase chain reaction (PCR) methods usinginfluenza virus natural genomic RNA isolated from the virus. For thepurpose of this application, nucleic acid sequence refers to RNA, DNA,cDNA or any synthetic variant thereof which encodes the protein.

Avian influenza A/Hong Kong/1073/99 (H9N2) virus was provided by Dr. K.Subbarao (Centers for Disease Control, Atlanta, Ga., USA). Viral genomicRNA was isolated by the acid phenol RNA extraction method underBiosafety Level 3 (BSL3) containment conditions at CDC using Trizol LSreagent (Invitrogen, Carlsbad, Calif. USA). cDNA molecules of the viralRNAs were obtained by reverse transcription using MuLV reversetranscriptase (InVitrogen) and PCR using oligonucleotide primersspecific for HA, NA, and M1 proteins and Taq I DNA polymerase(InVitrogen) (Table 1). The PCR fragments were cloned into the bacterialsubcloning vector, pCR2.1TOPO (InVitrogen), between Eco RI sites thatresulted in three recombinant plasmids, containing the HA, NA, and M1cDNA clones.

Example 3 RT-PCR Cloning of Human Influenza A/Sydney/5/94 (H3N2) ViralGenes

Influenza A/Sydney/5/94 (H3N2) virus was obtained from Dr. M. Massare(Novavax, Inc., Rockville, Md.). Viral genomic RNA was isolated by theRNA acid phenol extraction method under BSL2 containment conditions atNovavax, Inc. using Trizol LS reagent (Invitrogen). cDNA molecules ofthe viral RNAs were obtained by reverse transcription and PCR usingoligonucleotide primers specific for HA, NA, M1, M2, and NP proteins(Table 2). The PCR fragments were cloned into the bacterial subcloningvector, pCR2.1TOPO, between Eco RI sites that resulted in fiverecombinant plasmids, containing the HA, NA, M1, M2, and NP cDNA clones.

Example 4 Cloning of Avian Influenza A/Hong Kong/1073/99 Viral cDNAsinto Baculovirus Transfer Vectors

From the pCR2.1TOPO-based plasmids, the HA, NA, or M1 genes weresubcloned into pFastBac1 baculovirus transfer vector (InVitrogen) withinthe polyhedron locus and Tn7 att sites and downstream of the baculoviruspolyhedrin promoter and upstream of the polyadenylation signal sequence.The viral genes were ligated with T4 DNA ligase. For the HA gene, a BamHI-Kpn I DNA fragment from pCR2.1TOPO-HA was inserted into Bam HI-Kpn Idigested pFastBac1 plasmid DNA. For the NA gene, an Eco RI DNA fragmentfrom pCR2.1TOPO-NA was inserted into Eco RI digested pFastBac1 plasmidDNA. For the M1 gene, an Eco RI DNA fragment from pCR2.1TOPO-M1 wasinserted into Eco RI digested pFastBac1 plasmid DNA. Competent E. coliDH5α₌bacteria (InVitrogen) were transformed with these DNA ligationreactions, transformed colonies resulted, and bacterial clones isolated.The resulting pFastBac1-based plasmids, pFastBac1-HA, pFastBac1-NA, andpFastBac1-M1 were characterized by restriction enzyme mapping on agarosegels (FIG. 4A). The nucleotide sequences as shown on FIGS. 1-3 of thecloned genes were determined by automated DNA sequencing. DNA sequenceanalysis showed that the cloned influenza HA, NA, and M1 genes wereidentical to the nucleotide sequences for these genes as publishedpreviously [NA, HA, and M1 genes of influenza A/Hong Kong/1073/99 (H9N2)(GenBank accession numbers AJ404629, AJ404626, and AJ278646,respectively)].

Example 5 Cloning of Human Influenza A/Sydney/5/94 Viral cDNAs intoBaculovirus Transfer Vectors

From the pCR2.1TOPO-based plasmids, the HA, NA, M1, M2, and NP geneswere subcloned into pFastBac1 baculovirus transfer vector within thepolyhedron locus and Tn7 att sites and downstream of the baculoviruspolyhedrin promoter and upstream of the polyadenylation signal sequence.The viral genes were ligated with T4 DNA ligase. For the HA gene, a BamHI-Kpn I DNA fragment from pCR2.1TOPO-hHA3 was inserted into Bam HI-KpnI digested pFastBac1 plasmid DNA. For the NA gene, an Eco RI DNAfragment from pCR2.1TOPO-hNA was inserted into Eco RI digested pFastBac1plasmid DNA. For the M1 gene, an Eco RI DNA fragment from pCR2.1TOPO-hM1was inserted into Eco RI digested pFastBac1 plasmid DNA. For the M2gene, an Eco RI DNA fragment from pCR2.1TOPO-hM2 was inserted into EcoRI digested pFastBac1 plasmid DNA. For the NP gene, an Eco RI DNAfragment from pCR2.1TOPO-hNP was inserted into Eco RI digested pFastBac1plasmid DNA. Competent E. coli DH5α₌bacteria were transformed with theseDNA ligation reactions, transformed colonies resulted, and bacterialclones isolated. The resulting pFastBac1-based plasmids, pFastBac1-hHA3,pFastBac1-hNA2, pFastBac1-hM1, pFASTBAC1-hM2, and pFASTBAC1-hNP werecharacterized by restriction enzyme mapping on agarose gels. Thenucleotide sequences of the cloned genes were determined by automatedDNA sequencing. DNA sequence analysis showed that the cloned influenzaHA, NA, M1, M2, and NP genes were identical to the nucleotide sequencesfor these genes as published previously.

Example 6 Construction of Multigenic Baculovirus Transfer VectorsEncoding Multiple Avian Influenza A/Hong Kong/1073/99 Viral Genes

In order to construct pFastBac1-based bacmid transfer vectors expressingmultiple influenza A/Hong Kong/1073/99 (H9N2) virus genes, initially aSna BI-Hpa I DNA fragment from pFastBac1-M1 plasmid containing the M1gene was cloned into Hpa I site of pFastBac1-HA. This resulted inpFastBac1-HAM plasmid encoding both HA and M1 genes within independentexpression cassettes and expressed under the control of separatepolyhedrin promoters.

Finally, a Sna BI-Avr II DNA fragment from pFastBac1-HAM containing theHA and M1 expression cassettes, was transferred into Hpa I-Avr IIdigested pFastBac1-NA plasmid DNA. This resulted in the plasmidpFastBac1-NAHAM encoding three independent expression cassettes forexpression of influenza HA, NA, and M1 genes and expressed under thecontrol of separate polyhedrin promoters (FIG. 4B).

In another example, the H3 gene from pFASTBAC1-hHA3 (see Example 5) wascloned into pFASTBAC1-NAHAM as a fourth influenza viral gene for theexpression and production of heterotypic influenza VLPs.

Example 7 Generation of Multigenic Recombinant Baculovirus Encoding NA,HA, and M1 Genes of Avian Influenza A/Hong Kong/1073/99 Virus in InsectCells

The resulting multigenic bacmid transfer vector pFastBac1-NAHAM was usedto generate a multigenic recombinant baculovirus encoding avianinfluenza A/Hong Kong/1073/99 (H9N2) HA, NA, and M1 genes for expressionin insect cells. Recombinant bacmid DNAs were produced by site-specificrecombination at polyhedrin and Tn7 att DNA sequences betweenpFastBac1-NAHAM DNA and the AcMNPC baculovirus genome harbored incompetent E. coli DH10BAC cells (InVitrogen) (FIG. 4B). Recombinantbacmid DNA was isolated by the mini-prep plasmid DNA method andtransfected into Sf-9s cells using the cationic lipid CELLFECTIN(InVitrogen). Following transfection, recombinant baculoviruses wereisolated, plaque purified, and amplified in Sf-9S insect cells. Virusstocks were prepared in Sf-9S insect cells and characterized forexpression of avian influenza viral HA, NA, and M1 gene products. Theresulting recombinant baculovirus was designated bNAHAM-H9N2.

Example 8 Expression of Recombinant Avian Influenza a/Hong Kong/1073/99Proteins in Insect Cells

Sf-9S insect cells maintained as suspension cultures in shaker flasks at28° C. in serum-free medium (HyQ SFM, HyClone, Ogden, Utah) wereinfected at a cell density of 2×10⁶ cells/ml with the recombinantbaculovirus, bNAHAM-H9N2, at a Multiplicity of infection (MOI) of 3pfu/cell. The virus infection proceeded for 72 hrs. to allow expressionof influenza proteins. Expression of avian influenza A/Hong Kong/1073/99(H9N2) HA and M1 proteins in infected insect cells was confirmed bySDS-PAGE and Western immunoblot analyses. SDS-PAGE analysis wasperformed on 4-12% linear gradient NuPAGE gels (Invitrogen) underreduced and denaturing conditions. Primary antibodies in Westernimmunoblot analysis were polyclonal rabbit antiserum raised againstavian influenza A/Hong Kong/1073/99 (H9N2) obtained from CDC andmonoclonal murine antiserum to influenza M1 protein (Serotec, UK).Secondary antibodies for Western immunoblot analysis were alkalinephosphatase conjugated goat IgG antisera raised against rabbit or mouseIgG (H+L) (Kirkegaard and Perry Laboratories, Gaithersburg, Md., USA).Results of these analyses (FIG. 5) indicated that the HA and M1 proteinswere expressed in the baculovirus-infected insect cells.

Example 9 Purification of Recombinant Avian Influenza H9N2 Virus-LikeParticles and Macromolecular Protein Complexes

Culture supernatants (200 ml) from Sf-9S insect cells infected with therecombinant baculovirus bNAHAM-H9N2 that expressed avian influenzaA/Hong Kong/1073/99 (H9N2) HA, NA, and M1 gene products were harvestedby low speed centrifugation. Culture supernatants were clarified bycentrifugation in a Sorval RC-5B superspeed centrifuge for 1 hr at10,000×g and 4° C. using a GS-3 rotor. Virus and VLPs were isolated fromclarified culture supernatants by centrifugation in a Sorval OTD-65ultracentrifuge for 3 hr at 27,000 rpm and 4° C. using a Sorval TH-641swinging bucket rotor. The virus pellet was resuspended in 1 ml of PBS(pH 7.2), loaded onto a 20-60% (w/v) discontinuous sucrose stepgradient, and resolved by centrifugation in a Sorval OTD-65ultracentrifuge for 16 hr at 27,000 rpm and 4° C. using a Sorval TH-641rotor. Fractions (0.5 ml) were collected from the top of the sucrosegradient.

Influenza proteins in the sucrose gradient fractions were analyzed bySDS-PAGE and Western immunoblot analyses as described above in Example6. The HA and M1 proteins were found in the same sucrose gradientfractions (FIG. 6) as shown by Western blot analysis and suggested thatthe HA and M1 proteins were associated as macromolecular proteincomplexes. Also the HA and M1 proteins were found in fractionsthroughout the sucrose gradient suggesting that these recombinant viralproteins were associated with macromolecular protein complexes ofdifferent densities and compositions.

Example 10 Analysis of Recombinant Avian Influenza H9N2 VLPs andProteins by Gel Filtration Chromatography

Protein macromolecules such as VLPs and monomeric proteins migratedifferently on gel filtration or size exclusion chromatographic columnsbased on their mass size and shape. To determine whether the recombinantinfluenza proteins from sucrose gradient fractions were monomericproteins or macromolecular protein complexes such as VLPs, achromatography column (7 mm×140 mm) with a resin bed volume of 14 ml ofSepharose CL-4B (Amersham) was prepared. The size exclusion column wasequilibrated with PBS and calibrated with Dextran Blue 2000, DextranYellow, and Vitamin B12 (Amersham Pharmacia) with apparent molecularweights of 2,000,000; 20,000; and 1,357, respectively, to ascertain thecolumn void volume. Dextran Blue 2000 eluted from the column in the voidvolume (6 ml fraction). As expected, the recombinant influenza proteincomplexes eluted from the column in the void volume (6 ml fraction)also. This result was characteristic of a high molecular weightmacromolecular protein complex such as VLPs. Viral proteins in thecolumn fractions were detected by Western immunoblot analysis asdescribed above in Example 6. The M1 proteins were detected in the voidvolume fractions (FIG. 7). As expected baculovirus proteins were also inthe void volume.

Example 11 Electron Microscopy of Recombinant Influenza VLPs

To determine whether the macromolecular protein complexes isolated onsucrose gradients and containing recombinant avian influenza proteinshad morphologies similar to influenza virions, electron microscopicexamination of negatively stained samples was performed. Recombinantavian influenza A/Hong Kong/1073/99 (H9N2) protein complexes wereconcentrated and purified from culture supernatants byultracentrifugation on discontinuous sucrose gradients as described inExample 7. Aliquots of the sucrose gradient fractions were treated witha 2% glutaraldehyde in PBS, pH7.2, absorbed onto fresh dischargedplastic/carbon-coated grids, and washed with distilled water. Thesamples were stained with 2% sodium phosphotungstate, pH 6.5, andobserved using a transmission electron microscope (Philips). Electronmicrographs of negatively-stained samples of recombinant avian influenzaH9N2 protein complexes from two sucrose gradient fractions showedspherical and rod-shaped particles (FIG. 8) from two sucrose gradientfractions. The particles had different sizes (60 and 80 nm) andmorphologies. Larger complexes of both types of particles were alsodetected, as well as rod-shaped particles (FIG. 8). All observed proteincomplex structures exhibited spike like surface projections resemblinginfluenza virus HA and NA peplomers. Since the size and appearance ofthe 80 nm particles was similar to that of wild type influenza virusparticles, these structures likely represented enveloped influenza VLPs.The smaller particles of approximately 60 nm probably representedsubviral particles that differed from the above VLPs bothmorphologically and structurally.

Example 12 Analysis of Functional Characteristics of Influenza Proteinsby Hemagglutination Assay

To determine whether the purified influenza VLPs and proteins possessedfunctional activities, such as hemagglutination and neuraminidaseactivity, which were characteristic for influenza virus, the purifiedinfluenza VLPs and proteins were tested in hemagglutination andneuraminidase assays.

For the hemagglutination assay, a series of 2-fold dilutions of sucrosegradient fractions containing influenza VLPs or positive control wildtype influenza virus type A were prepared. Then they were mixed with0.6% guinea pig red blood cells in PBS (pH 7.2) and incubated at 4° C.for 1 to 16 hr. As a negative control, PBS was used. The extent ofhemagglutination was determined visually, and the highest dilution offraction capable of agglutinating guinea pig red blood cells wasdetermined (FIG. 9). The highest hemagglutination titer observed for thepurified influenza VLPs and proteins was 1:4000, which was higher thanthe titer shown by the wild type influenza control, which was 1:2000.

Example 13 Analysis of Functional Characteristics of Influenza Proteinsby Neuraminidase Assay

The amount of neuraminidase activity in influenza VLP-containing sucrosegradient fractions was determined by the neuraminidase assay. In thisassay the NA (an enzyme) acted on the substrate (fetuin) and releasedsialic acid. Arsenite reagent was added to stop enzyme activity. Theamount of sialic acid liberated was determined chemically with thethiobarbituric acid that produced a pink color in proportion to freesialic acid. The amount of color (chromophor) was measured in aspectrophotometer at wavelength 594 nm. The data, as depicted in FIG. 8,showed that a significant amount of sialic acid was produced byVLP-containing fractions of the sucrose gradients and that thesefractions corresponded to those fractions exhibiting hemagglutinationactivity.

Example 13 Immunization of BALB/c Mice with Functional HomotypicRecombinant Influenza H9N2 VLPs

The immunogenicity of the recombinant influenza VLPs was ascertained byimmunization of mice followed by Western blot analysis of immune sera.Recombinant VLPs (1 μg/injection) comprised of viral HA, NA, and M1proteins from avian influenza virus type A/Honk Kong/1073/99 andpurified on sucrose gradients were inoculated subcutaneously into thedeltoid region of ten (10) female BALB/c mice at day 0 and day 28 (FIG.11). PBS (pH 7.2) was administered similarly as a negative control intofive (5) mice. The mice were bled from the supraorbital cavity at day-1(pre-bleed), day 27 (primary bleed), and day 54 (secondary bleed). Serawere collected from blood samples following overnight clotting andcentrifugation.

For Western blot analysis, 200 ng of inactivated avian influenza virustype A H9N2 or cold-adapted avian influenza virus type A H9N2, as wellas See Blue Plus 2 pre-stained protein standards (InVitrogen), wasdenatured (95° C., 5 minutes) and subjected to electrophoresis underreduced conditions (10 mM β-mercaptoethanol) on 4-12% polyacrylamidegradient NuPAGE gels (InVitrogen) in MES buffer at 172 volts until thebromophenol blue tracking dye disappeared. For protein gels, theelectrophoresced proteins were visualized by staining with ColloidalCoomassie Blue reagent (InVitrogen). Proteins were transferred from thegel to nitrocellulose membranes in methanol by the standard Western blotprocedure. Sera from VLP-immunized mice and rabbits immunized withinactivated avian influenza virus H9N2 (positive control sera) werediluted 1:25 and 1:100, respectively, in PBS solution (pH 7.2) and usedas primary antibody. Protein bound membranes, which were blocked with 5%casein, were reacted with primary antisera for 60 minutes at roomtemperature with constant shaking. Following washing of primary antibodymembranes with phosphate buffered saline solution containing Tween 20,secondary antisera [goat anti-murine IgG-alkaline phosphatase conjugate(1:10,000) or goat anti-rabbit IgG-alkaline phosphatase conjugate(1:10,000)] were reacted 60 minutes with the membrane. Following washingof secondary antibody membranes with phosphate buffered saline solutioncontaining Tween 20, antibody-binding proteins on the membranes werevisualized by development with the chromogenic substrate such asNBT/BCIP (InVitrogen).

The results of Western blot analysis (FIG. 12) were that proteins withmolecular weights similar to viral HA and M1 proteins (75 and 30 kd,respectively) bound to positive control sera (FIG. 12B) and sera frommice immunized with the recombinant influenza H9N2 VLPs (FIG. 12A).These results indicated that the recombinant influenza H9N2 VLPs alonewere immunogenic in mice by this route of administration.

The following references are incorporated herein by reference:

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Other Embodiments

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims:

TABLE 1 Fraction#* Titer  1 <1:500  3 <1:500  5   1:500  7   1:1000  9  1:2000 11   1:2000 12   1:4000 14   1:500 16 <1:500 PBS** <1:500A/Shangdong/9/93***   1:1000 *Fractions from 20-60% sucrose gradient**Negative control ***Positive control

TABLE 2 TABLE 2 Virus Strain Gene RT-PCR Primer Type  Sydney/ Hemag-Forward 5′-A GGATCCATGAA A 5/97 glutinin GACTATCATTGCTTTGAG-3′ (H3N2)(HA) (SEQ ID NO: 4) Reverse 5′-A GGTACCTCAAAT GCAAATGTTGCACCTAATG-3′(SEQ ID NO: 5) Neura- Forward 5′-GGGGACAAGTTTGT minidaseACAAAAAAGCAGGCTTAGAAGGAGA (NA) TAGAACC ATG AATCCAAATCAAA AGATAATAAC-3′(SEQ ID NO: 6) Reverse 5′-GGGGACCACTTTGT ACAAGAAAGCTGGGTCCTATATAGGCATGAGATTGATGTCCGC-3′ (SEQ ID NO: 7) Matrix Forward 5′-AAA GAATTC ATG(MI) AGTCTTCTAACCGAGGTCGAAAC GTA-3′(SEQ ID NO: 8)Reverse 5′-AAA TTCGAA TTA CTCCAGCTCTATGCTGAGAAAAT GAC-3′(SEQ. ID NO: 9)M2 Forward 5′-A GAATTC ATG AG TCTTCTAACCGAGGTCGAAACGCCTATCAGAAACGAATGGGGGTGC-3′ (SEQ ID NO: 10) Reverse 5′-AAA TTCGAA TTACTCCAGCTCTATGCTGACAAAAT GAC-3′(SEQ ID NO: 11) Nucleo-Forward 5′-A GAATTC ATG GC protein GTCCCAAGGCACCAACG-3′ (NP)(SEQ ID NO: 12) Reverse 5′-A GCGGCCGCTTAAT TGTCGTACTCCTCTGCATTGTCTCCGAAGAAATAAG-3′(SEQ ID NO: 13) Type Harbin Hemag-Forward 5′-A GAATTC ATG AA B glutinin GGCAATAATTGTACTACTCATGG-3′ (HA)(SEQ ID NO: 14) Reverse 5′-A GCGGCCGCTTATA GACAGATGGAGCAAGAAACATTGTCTCTGGAGA-3′(SEQ ID NO: 15) Neura- Forward 5′-A GAATT CATG CT minidaseACCTTCAACTATACAAACG-3′ (NA) (SEQ ID NO: 16) Reverse 5′-A GCGGCCGCTTACAGAGCCATATCAACACCTGTGAC AGTG-3′(SEQ ID NO: 17)

The invention claimed is:
 1. A method of preventing influenza in avertebrate comprising administering a vaccine to the vertebrate, whereinthe vaccine comprises: i) a virus-like particle (VLP) comprisinginfluenza structural proteins, wherein the influenza structural proteinsof the VLP consist of M1, HA, and NA, wherein the VLP is self-assembledin a host cell from a recombinant construct wherein the M1 protein isfrom an avian influenza virus, and wherein the M1 protein is from adifferent strain of influenza virus than the influenza HA protein andthe influenza NA protein; and ii) a carrier or diluent.
 2. The method ofclaim 1 wherein the vaccine is a multivalent vaccine.
 3. The method ofclaim 1, wherein the host cell is a eukaryotic cell.
 4. The method ofclaim 3, wherein the eukaryotic cell is an insect cell.
 5. The method ofclaim 3, wherein the recombinant construct is a recombinant baculovirusconstruct.
 6. The method of claim 1, wherein the HA and NA proteins arederived from a mammalian influenza strain.
 7. The method of claim 1,wherein the HA and NA proteins are derived from the group consisting ofsubtype A and B influenza viruses.
 8. The method of claim 1, furthercomprising an adjuvant.
 9. The method of claim 1 wherein the HA proteinexhibits hemagglutinin activity.
 10. The method of claim 1, wherein theNA protein exhibits neuraminidase activity.